OVERVIEW The Leica DM-RXA is a fully computer-controlled light microscope. The DAR is collaborating with Dr. Mueller's lab to design and test a software interface which will allow 3D time-lapse (4D) studies to be conducted using this microscope. The bulk of this project will involve Mr. Thomas writing, testing and debugging computer code as well as doing real-world testing of data collection using the Leica DM-RXA microscope itself. Dr. Fleischmann will then be involved in testing and debugging the software as well as in pursuing real-world biological applications of the software in his lab in Switzerland. This project is divided into two parts. STAGE 1: We intend to develog both a C-based stand-alone and an NIH-Image dependent software interface to allow the DM-RXA microscope to function in the collection of basic 4-D data sets using high-resolution Nomarski DIC optics. This includes the orchestration of illumination shutters, stage motors and digitizing video fi-amegrabbers from a Macintosh PPC computer as outlined in Thomas et al., (1996) Science, 273, 603-607. Easy-to-use and intuitive graphic interfaces should allow this software to be uncomplicated and permit a low learning curve for researchers hoping to become involved in 4D research. The software should also be made available free of charge via the internet to researchers around the world. NOTE This phase of the project has been completed successfully. STAGE 2: This phase of the project will investigate the possibility of gathering simultaneous (or nearly simultaneous) DIC and fluorescent 4D data sets from the same sample. BIOLOGICAL RESEARCH PLANS: The biological research plans for each phase of this project are as follows: Stage I - determination of the lineage of newly isolated mutant embryos. Stage 2: Expression studies with strains carrying GFP reporter constructs. Our main focus lies on the ceh-13 gene, but it could be used for any gene. First we will determine the wild type expression pattern. Then we will analyze mutants (which we think differ from the wild VyW expression pattern) and strains carrying deletions in the promoter region of the GFP reporter construct.